Diaphragm muscle fiber weakness and ubiquitin-proteasome activation in critically ill patients.

RATIONALE: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity and duration of hospital stay. To date, the nature of diaphragm weakness and its underlying pathophysiologic mechanisms are poorly understood. OBJECTIVES: We hypothesized that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and that the ubiquitin-proteasome pathway is activated in the diaphragm. METHODS: We obtained diaphragm muscle biopsies from 22 critically ill patients who received mechanical ventilation before surgery and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (control subjects). In a proof-of-concept study in a muscle-specific ring finger protein-1 (MuRF-1) knockout mouse model, we evaluated the role of the ubiquitin-proteasome pathway in the development of contractile weakness during mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Both slow- and fast-twitch diaphragm muscle fibers of critically ill patients had approximately 25% smaller cross-sectional area, and had contractile force reduced by half or more. Markers of the ubiquitin-proteasome pathway were significantly up-regulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation. CONCLUSIONS: These findings show that diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness, and in the diaphragm of critically ill patients the ubiquitin-proteasome pathway is activated. This study provides rationale for the development of treatment strategies that target the contractility of diaphragm fibers to facilitate weaning.

Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C.

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2.

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency.

Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin's N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.

Selective diaphragm muscle weakness after contractile inactivity during thoracic surgery.

RATIONALE: Postoperative pulmonary complications are significant contributors to morbidity in patients who have undergone upper abdominal, thoracic, or cardiac surgery. The pathophysiology of these complications might involve postoperative inspiratory muscle weakness. The nature of postoperative inspiratory muscle weakness is unknown. OBJECTIVE: To investigate the effect of surgery on the functioning of the diaphragm, the main muscle of inspiration. METHODS: Serial biopsies from the diaphragm and the latissimus dorsi muscle were obtained from 6 patients during thoracotomy for resection of a tumor in the right lung. Biopsies were taken as soon as the diaphragm had been exposed (t(0)) and again after 2 hours (t(2)). The contractile performance of demembranated muscle fibers, as well as fiber morphology and markers for proteolysis, was determined. RESULTS: In all patients, the force-generating capacity of diaphragm muscle fibers at t(2) was significantly reduced (~35%) compared with that at t(0), with a more pronounced force loss in type 2 fibers compared with type 1 fibers. Diaphragm weakness was not part of a generalized muscle weakness as contractile performance of latissimus dorsi fibers was preserved at t(2). Diaphragm fiber size and myofibrillar structure were not different at t(2) compared with t0, but myosin heavy chain type 2 was significantly reduced at t(2) and MuRF-1 mRNA and protein levels were elevated at t(2). CONCLUSIONS: Only 2 hours of thoracic surgery causes marked, and selective, diaphragm muscle fiber weakness.

COVID-19 Exposure Tracking Within Public Health & Safety Enterprises: Findings to Date & Opportunity for Further Research.

Where there is limited access to COVID-19 tests, or where the results of such tests have been delayed or even invalidated (e.g., California and Utah), there is a need for scalable alternative approaches-such as a heuristic model or "pregnancy test for COVID-19" that can factor in the time denominator (i.e., duration of symptoms). This paper asks whether infection among these public health and safety agencies is a "canary in the coal mine," litmus test, or microcosm (pick your analogy) for the communities in which they operate. Can COVID-19 infection counts and rates be seen "moving around" communities by examining the virus's effect on emergency responders themselves? The troubling question of emergency responders becoming "human indicator values" is relevant to maintaining the health of Mobile Medicine (EMS and Fire) personnel, as well as Police, who are an under-attended population, because these groups our collective resiliency would crash. It has further implications for policies regarding, and investments in, exposure tracking and contact tracing, PPE acquisition, and mental and physical wellness. DESIGN: We aggregated data from four (4) different EMS documentation systems across twelve (12) states using the MEDIVIEW BEACON Prehospital Health Information Exchange. We then outputted lists of charts containing critical ICD-10 values that had been identified by the WHO, the CDC, and the Los Angeles County Fire Deptartment's EMS Bureau as inclusion criteria for possible signs, symptoms, and clinical impressions of COVID-19 infection. RESULTS: Three important results emerged from this study: (1) a demonstration of frequent exposure to possible COVID-19 infection among Mobile Medical (EMS & Fire) care providers in the states whose data were included; (2) a demonstration of the nervousness of the general population, given that calls for help due to possible COVID-19 based on symptomology exceeded the number of responses with a correlating "provider impression" after an informed clinical assessment; and (3) the fact that this study was empowered by a public-private partnerships between a technology startup and numerous public health and public safety agencies, offers a template for success in rapidly implementing research and development collaborations. LIMITATIONS: This study incorporates data from only (a) twelve (12) states, and (b) four (4) Mobile Medical documentation systems. We sought to combat these limitations by ensuring that our sample crosses agencies types, geographies, population demographics, and municipal environments (i.e., rural vs. urban). CONCLUSIONS: Other studies have noted that EMS agencies are tasked with transporting the "sickest of the sick." We found that PPE is particularly essential where the frequency of encounters between potentially-or actually-infected patients is high, because from Los Angeles County to rural Texas, without sufficient protection, public health and public safety agencies have become microcosms of the communities they are meant to protect. Indeed, data from the first six months of the declared pandemic in the U.S.A. show that intra-departmental spread is one of (if not the) riskiest sources of infection among Mobile Medical professionals.

Modulation of muscle atrophy, fatigue and MLC phosphorylation by MuRF1 as indicated by hindlimb suspension studies on MuRF1-KO mice.

MuRF1 is a member of the TRIM/RBCC superfamily, a gene family that encompasses a large variety of proteins, all sharing the conserved TRIM (Tripartite Motive) sequential array of RING, B-box, and coiled-coil domains. Within this family, MuRF1(also named TRIM63) is a specialized member that contributes to the development of muscle atrophy and sarcopenia. Here we studied MuRF1's role in muscle atrophy during muscle unloading induced by hindlimb suspension. Consistent with previous studies, we found that MuRF1 inactivation leads to an attenuated muscle atrophy response. The amount of protection was higher as compared to the denervation model, and within the 10 day-suspension period the soleus muscle was spared from atrophy in MuRF1-KO mice. Contractility studies on hindlimb suspended muscle tissues suggested that MuRF1's functions extend beyond muscle trophicity and implicate MuRF1 in muscle fatigue and MLC phosphorylation control: soleus muscle from MuRF1-KO mice fatigued significantly faster and in addition showed a reduced posttetanic twitch potentiation. Thus the present work further established the role of MuRF1 in muscle atrophy and for the first time shows that MuRF1 plays a role in muscle fatigue and twitch potentiation.

Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle.

Previous work suggested that altered Ca(2+) homeostasis might contribute to dysfunction of nebulin-free muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-inhibitor sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as expression of other Ca(2+)-handling proteins. Ca(2+) uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca(2+) transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca(2+), as well as in electrically stimulated intact muscle. We found profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of other Ca(2+)-handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca(2+) uptake was >3-fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact myofibers. Ca(2+)-activated stress in skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent compared with wild type. Half-relaxation time was significantly longer in nebulin-free compared with wild-type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca(2+) handling of the SR-myofibrillar system.

Small-molecule-mediated chemical knock-down of MuRF1/MuRF2 and attenuation of diaphragm dysfunction in chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small-molecule inhibitor of MuRF1 in treating CHF-induced diaphragm myopathy and loss of contractile function. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham-operated animals (sham) served as controls. One week post-left anterior descending coronary artery ligation animals were randomized into two groups-one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946-a recently described MuRF1-interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed. RESULTS: Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up-regulation of the MuRF1 and MuRF2 was attenuated after infarct. CONCLUSIONS: Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.

Longitudinal transcriptome-wide gene expression analysis of sleep deprivation treatment shows involvement of circadian genes and immune pathways.

Therapeutic sleep deprivation (SD) rapidly induces robust, transient antidepressant effects in a large proportion of major mood disorder patients suffering from a depressive episode, but underlying biological factors remain poorly understood. Research suggests that these patients may have altered circadian molecular genetic 'clocks' and that SD functions through 'resetting' dysregulated genes; additional factors may be involved, warranting further investigation. Leveraging advances in microarray technology enabling the transcriptome-wide assessment of gene expression, this study aimed to examine gene expression changes accompanying SD and recovery sleep in patients suffering from an episode of depression. Patients (N = 78) and controls (N = 15) underwent SD, with blood taken at the same time of day before SD, after one night of SD and after recovery sleep. A transcriptome-wide gene-by-gene approach was used, with a targeted look also taken at circadian genes. Furthermore, gene set enrichment, and longitudinal gene set analyses including the time point after recovery sleep, were conducted. Circadian genes were significantly affected by SD, with patterns suggesting that molecular clocks of responders and non-responders, as well as patients and controls respond differently to chronobiologic stimuli. Notably, gene set analyses revealed a strong widespread effect of SD on pathways involved in immune function and inflammatory response, such as those involved in cytokine and especially in interleukin signalling. Longitudinal gene set analyses showed that in responders these pathways were upregulated after SD; in non-responders, little response was observed. Our findings emphasize the close relationship between circadian, immune and sleep systems and their link to etiology of depression at the transcriptomic level.

Titin expression in human articular cartilage and cultured chondrocytes: a novel component in articular cartilage biomechanical sensing?

In striated muscle tissues, the giant protein titin acts as a biomechanically active filament system, coupling stress/strain to gene expression. The objective of the study is to show the existence of titin fragments in human articular cartilage, as in diarthodial joints, chondrocytes are also known to sense and respond to stretching. We have surveyed human cultured cartilage collected from adults with osteoarthritis (OA), without OA and from infants with a set of titin antibodies and primer pairs. Three different antibodies were used for immunolabelling, reacting with titin's N-terminal Z1-Z2 domains, its Novex III exon, and with its PEVK region. An antibody directed to a titin ligand was included, since in cardiac muscle, this has been shown to participate in the transmission of stretch dependent titin-based signals. Our results indicate that although at low levels, titin is expressed in cartilage. Primer pairs detected titin transcripts in cartilage, and consistent with this, antibodies directed to titin's Z-disc region and to its elastic region stained cartilage. Moreover, we also could detect transcription of the titin ligand CARP. Components of the stretch dependent signal machinery in muscle are also expressed in cartilage. Further studies are warranted to address if common stress/strain dependent signalling are conserved in muscle and cartilage tissues.

Myocardial expression of Murf-1 and MAFbx after induction of chronic heart failure: Effect on myocardial contractility.

OBJECTIVE: In chronic heart failure (CHF) the myocardial expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), which is thought to contribute to myocardial remodeling, was found to be increased. However, it is unknown whether the E3-ubiquitin ligases MAFbx and Murf-1 are involved in this remodeling process and whether their expression is regulated by TNF-alpha. METHODS: Rats underwent ligation of the left coronary artery to induce CHF or were sham-operated. The expression of MAFbx/Murf-1 and troponin I was analyzed by RT-PCR and Western blotting in the non-infarcted area of the left ventricle. In cell culture experiments the potency of TNF-alpha to stimulate Murf-1/MAFbx expression, the intracellular signaling pathway, and the involvement of the E3-ligases for the impairment of contractility were assessed. RESULTS: In CHF the myocardial expression of TNF-alpha was elevated 3.1-fold as compared to control. This was associated with a 4.5-fold and 2.7-fold increase in MAFbx and Murf-1 expression, respectively. A positive correlation between TNF-alpha and the expression of MAFbx or Murf-1 was evident. In neonatal rat cardiomyocytes, TNF-alpha induced the expression of MAFbx through p38MAPK-dependent pathways, whereas the induction of Murf-1 required the activation of the p42/44 MAPK pathway. Exposure of cardiomyocytes to TNF-alpha resulted in troponin I ubiquitinylation, subsequent degradation, and a decline in contractility. This was completely abrogated by siRNAs against Murf-1/MAFbx. CONCLUSION: TNF-alpha, which is increasingly expressed in CHF, induces troponin I degradation through a MAFbx/Murf-1-dependent pathway. This was associated with an impairment of contractility and might be one mechanism involved in the adverse remodeling process in CHF.

MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination.

MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.

Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness.

BACKGROUND: Titin contains a molecular spring segment that underlies passive myocardial stiffness. Myocardium coexpresses titin isoforms with molecular spring length variants and, consequently, distinct stiffness characteristics: the stiff N2B isoform (short spring) and more compliant N2BA isoform (long spring). We tested whether changes in titin isoform expression occur in the diastolic dysfunction that accompanies heart failure. METHODS AND RESULTS: We used the tachycardia-induced dilated cardiomyopathy canine model (4-week pacing) and found that control myocardium coexpresses the N2B and N2BA isoforms at similar levels, whereas in dilated cardiomyopathy the expression ratio had shifted, without affecting the amount of total titin, toward more prominent N2B expression. This shift was accompanied by elevated titin-based passive muscle stiffness. Pacing also resulted in significant upregulation of obscurin, an approximately 800-kDa elastic protein with several signaling domains. CONCLUSIONS: Coexpression of titin isoforms with distinct mechanical properties allows modulation of passive stiffness via adjustment of the isoform expression ratio. The canine pacing-induced heart failure model uses this mechanism to increase myocardial stiffness. Thus, changes in titin isoform expression may play a role in diastolic dysfunction in heart failure.

Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy.

BACKGROUND: The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. METHODS AND RESULTS: SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (P<0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (n=7), intermediate in those with pseudonormal filling (n=6), and lowest in the group with restrictive filling (n=7). Mechanical measurements on LV muscle strips dissected from these hearts (n=8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O2 consumption (n=10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). CONCLUSIONS: Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.

The complete mouse nebulin gene sequence and the identification of cardiac nebulin.

Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle.

The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.

Induction and myofibrillar targeting of CARP, and suppression of the Nkx2.5 pathway in the MDM mouse with impaired titin-based signaling.

Muscular dystrophy with myositis (mdm) is a recessive mouse mutation that is caused by a small deletion in the giant elastic muscle protein titin. Homozygous mdm/mdm mice develop a progressive muscular dystrophy, leading to death at approximately 2 months of age. We surveyed the transcriptomes of skeletal muscles from 24 day old homozygous mdm/mdm and +/+ wild-type mice, an age when MDM animals have normal passive and active tensions and sarcomeric structure. Of the 12488 genes surveyed (U74 affymetrix array), 75 genes were twofold to 30-fold differentially expressed, including CARP (cardiac ankyrin repeat protein), ankrd2/Arpp (a CARP-like protein) and MLP (muscle LIM protein), all of which associate with the titin filament system. The four genes most strongly affected (eightfold to 30-fold change) were all members of the CARP-regulated Nkx-2.5-dependent signal pathway, and CARP mRNA level was 30-fold elevated in MDM skeletal muscle tissues. The CARP protein overexpressed in MDM became associated with the I-band region of the sarcomere. The mdm mutation excises the C-terminal portion of titin's N2A region, abolishing its interaction with p94/calpain-3 protease. Thus, the composition of the titin N2A protein complex is altered in MDM by incorporation of CARP and loss of p94/calpain-3. These changes were absent from the following control tissues (1). cardiac muscles from homozygous mdm/mdm animals, (2). skeletal and cardiac muscle from heterozygous mdm/+ animals, and (3). dystrophic muscles from MDX mice. Thus, the altered composition of the titin N2A complex is specific for the titin-based skeletal muscular dystrophy in MDM.

Induction of Ankrd1 in Dilated Cardiomyopathy Correlates with the Heart Failure Progression.

Progression of idiopathic dilated cardiomyopathy (IDCM) is marked with extensive left ventricular remodeling whose clinical manifestations and molecular basis are poorly understood. We aimed to evaluate the clinical potential of titin ligands in monitoring progression of cardiac remodeling associated with end-stage IDCM. Expression patterns of 8 mechanoptotic machinery-associated titin ligands (ANKRD1, ANKRD2, TRIM63, TRIM55, NBR1, MLP, FHL2, and TCAP) were quantitated in endomyocardial biopsies from 25 patients with advanced IDCM. When comparing NYHA disease stages, elevated ANKRD1 expression levels marked transition from NYHA < IV to NYHA IV. ANKRD1 expression levels closely correlated with systolic strain depression and short E wave deceleration time, as determined by echocardiography. On molecular level, myocardial ANKRD1 and serum adiponectin correlated with low BAX/BCL-2 ratios, indicative of antiapoptotic tissue propensity observed during the worsening of heart failure. ANKRD1 is a potential marker for cardiac remodeling and disease progression in IDCM. ANKRD1 expression correlated with reduced cardiac contractility and compliance. The association of ANKRD1 with antiapoptotic response suggests its role as myocyte survival factor during late stage heart disease, warranting further studies on ANKRD1 during end-stage heart failure.

Comparison of gene expression profiles in the blood, hippocampus and prefrontal cortex of rats.

BACKGROUND: The comparability of gene expression between blood and brain tissues is a central issue in neuropsychiatric research where the analysis of molecular mechanisms in the brain is of high importance for the understanding of the diseases and the discovery of biomarkers. However, the accessibility of brain tissue is limited. Therefore, knowledge about how easily accessible peripheral tissue, e. g. blood, is comparable to and reflects gene expression of brain regions will help to advance neuropsychiatric research. DESCRIPTION: Gene expression in the blood, hippocampus (HC) and prefrontal cortex (PFC) of genetically identical rats was compared using a genome-wide Affymetrix gene expression microarray covering 29,215 expressed genes. A total of 56.8% of 15,717 expressed genes were co-expressed in blood and at least one brain tissue, while 55.3% of all genes were co-expressed in all three tissues simultaneously. The overlapping genes included a set of genes of relevance to neuropsychiatric diseases, in particular bipolar disorder, schizophrenia and alcohol addiction. These genes included CLOCK, COMT, FAAH, NPY, NR3C1, NRGN, PBRM1, TCF4, and SYNE. CONCLUSIONS: This study provides baseline data on absolute gene expression and differences between gene expression in the blood, HC and PFC brain tissue of genetically identical rats. The present data represents a valuable resource for future studies as it might be used for first information on gene expression levels of genes of interest in blood and brain under baseline conditions. Limitations of our study comprise possible contamination of brain tissue with blood and the non-detection of genes with very low expression levels. Genes that are more highly expressed in the brain than in the blood are of particular interest since changes in their expression, e.g. due to disease status, or treatment, are likely to be detected in an experiment. In contrast, genes with higher expression in the blood than in the brain are less informative since their higher baseline levels could superimpose variation in brain.